This invention relates to methods for the specific depletion of activated T-lymphocytes particularly those belonging to the CD4+ subclass. Such activated T-lymphocytes e.g. CD4+ T-lymphocytes, are implicated in a number of conditions in humans including multiple sclerosis and transplant rejection. In particular, this invention provides a treatment in which activated T-lymphocytes e.g. CD4+ T-cells involved in a particular disease or condition are depleted while the non-activated T-lymphocyte e.g. CD4+ T-cells repertoire is unaffected.
The CD4+ T-lymphocyte (herein referred to as the CD4+ T-cell) is the central player in the immune system because of the xe2x80x9chelpxe2x80x9d it provides to other leukocytes in fighting off infection and potential cancerous cells. CD4+ T-cells play essential roles in both humeral and cell-mediated immunity and additionally they act during parasite infection to promote the differentiation of eosinophils and mast cells. If the CD4+ T-cell population is depleted (as is the case in AIDS patients) the host is rendered susceptible to a number of pathogens and tumours that do not ordinarily pose a threat to the host.
While CD4+ T-cells thus play an important beneficial role in disease prevention, the aberrant function of these cells can produce serious problems. In some individuals, the aberrant function of CD4+ T-cells leads to autoimmunity and other disease states (Swanborg, R. H., 1984; Cush, J. J., and Lipsky, P. E., 1988; Caspi et al., 1988). Autoimmune diseases in which CD4+ T-cells have been implicated include multiple sclerosis, rheumatoid arthritis and autoimmune uveitis (see generally, Steinman, L., 1993). In essence these diseases involve an aberrant immune response in which the immune system is subverted from its normal role of attacking invading pathogens and instead attacks the host body tissues, leading to illness and even death. The targeted host tissues vary between autoimmune diseases, for example, in multiple sclerosis the immune system attacks the white matter of the brain and spinal cord, in rheumatoid arthritis the immune system attacks the synovial lining of the joints. Activated CD4+ T-cells have also been implicated in other illnesses, including rejection of transplant tissues and organs and in the development of CD4+ T-cell lymphomas.
Investigations into conditions caused by aberrant CD4+ T-cell activity are focused on several animal models, and in particular on a number of experimentally induced autoimmune diseases. Research on these experimentally induced diseases in animals is premised on the idea that they will provide information useful in the treatment of the corresponding human diseases. In pursuit of this goal, it has been shown that CD4+ T-cells are responsible for several experimentally induced autoimmune diseases in animals, including experimental autoimmune endephalomyelitis (EAE), collagen induced arthritis (CIA), and experimental autoimmune uveitis (EAU).
EAE is induced by autoimmunizing animals against myelin basic protein (MBP, a component of the white matter of the brain and the spinal cord) and produces the same clinical symptoms observed in multiple sclerosis: demyelination and paralysis. Proof of the value of the EAE model as a comparative model for multiple sclerosis has been provided by evidence showing that these conditions share a causative nexus: Steinman and co-workers showed that the predominant cell type found in the brain lesions of multiple sclerosis patients is CD4+ T-cells (Oksenberg, J. R., et al., 1990) and that the T-cell receptor (the molecule responsible for antigen recognition) associated with the cells in these brain lesions had the same 3 amino acid binding motif for antigen recognition as on the CD4+ T-cells responsible for causing experimental autoimmune. encephalomyelitis (EAE) (Oksenberg, J. R., et al., 1993). All the evidence thus suggests that the EAE model will be useful in testing therapies for multiple sclerosis.
Research on a number of the experimentally induced autoimmune diseases, including EAE, CIA and EAU, has shown that antibodies that bind CD4+ T-cells when injected in vivo can inhibit the development of these diseases as well as inhibit transplantation rejection (Swanborg, R. H. 1983; Cobbold, S. P. et al., 1984; Steinman, L, 1993). This antibody-mediated effect depletes or inactivates all CD4+ cells in the body (the antibodies that bind to the CD4+ cells presumably block the activity of the cells and also target the CD4+ cells for destruction by the immune system.) This strategy has shown some success with rheumatoid arthritis and is now being tested for multiple sclerosis (see generally, Steinman, L., 1993).
While it appears that therapeutic approaches that destroy the CD4+ T-lymphocyte population might be effective in ameliorating these autoimmune diseases, this approach has one very major drawback. The treatment not only inhibits the function of those CD4+ T-cells that are antigen reactive and thus involved in the autoimmune disease process, but also the CD4+ T-cells that are quiescent and not involved in the disease. Since CD4+ T-cells are important in the general immune response (protecting the body against infectious agents), destruction of the entire CD4+ T-cell population leaves the patient severely immunocompromised and hence highly susceptible to infection. A preferable approach would be to remove only those CD4+ T-cells that are actively involved in the auto-immune response, leaving the remaining CD4+ T-cell population available for their normal role in the immune system.
This method of treatment has not yet been achieved. It is therefore an object of the present invention to provide a method of specifically depleting the population of activated CD4+ T-cells in a patient without affecting the quiescent CD4+ T-cell population.
In one aspect the present invention provides a method by which autoantigen specific T-cells, especially for example activated CD4+ T-cells, can be specifically eliminated in vivo, while leaving the quiescent population of T-cells especially for example unactivated CD4+ T-cells intact. This invention therefore provides a treatment useful for T-cell mediated especially for example activated CD4+ T-cell mediated autoimmune diseases such as multiple sclerosis, rheumatoid arthritis, sarcoidosis and autoimmune uveitis, graft versus host disease (GVHD) and/or inflammatory bowel disease. This invention also provides a method for eliminating other undesired immune responses caused by activated T-cells especially for example activated CD4+ T-cells such as rejection of transplanted tissue and organs in transplant recipients. Furthermore, the present invention provides a method of specifically eliminating activated CD4+ T-cell lymphomas from the body. The present invention also provides a method for early diagnosis of conditions mediated by activated T-cells especially for example activated CD4+ T-cells by detecting the presence of autoreactive T-cells at the site of autoimmune lesions and potentially harmful T-cell lymphomas. This early diagnosis provides an indication that the methods of treatment provided by the present invention may be effective and can facilitate earlier treatment of the condition than might otherwise be possible.
The present invention is also based on the discovery that a particular protein antigen, termed OX-40 (herein referred to as the OX-40 antigen), is specifically expressed on the cell surface of antigen activated T-cells especially for example activated CD4+ T-cells. In particular, using the EAE disease model in rats, this antigen was shown to be expressed on the surface of activated autoantigen-specific CD4+ T-cells present at the site of inflammation (the spinal cord in this disease model) but absent on CD4+ T-cells at non-inflammatory sites. Furthermore, the highest expression of this antigen on these CD4+ T-cells was found to occur on the day prior to initiation of clinical signs of autoimmunity; the expression of this antigen decreased as the disease progressed. The specificity of expression of the OX-40 antigen and the transient nature of this expression, shown for the first time in the present invention, motivated the testing of this antigen as a possible target for antibody mediated depletion of activated T-cells in animals such as humans with T-cell mediated conditions.
The applicants have cloned and sequenced the cDNA encoding the OX-40 antigen. Thus the invention provides a nucleic acid having the sequence shown in Seq ID No 1 or a sub-sequence of SEQ ID No 1 which encodes an antigenic polypeptide; or a variant or allele thereof; or a complementary strand to any of these. A particular sub-sequence of SEQ ID No 1 comprises nucleotide bases 15 to 848 shown in Seq ID No 1 or its complementary strand.
Further according to the invention there is provided a polypeptide comprising an amino acid sequence encoded by a nucleic acid as described above or a derivative thereof. Suitably the polypeptide comprises an amino acid sequence encoded by a sub-sequence of the sequence shown in SEQ ID No 1 and which includes an antigenic determinant.
Among the variants of nucleic acid sequences and polypeptides contemplated by the invention are (for example) DNA sequence variants importing no change in encoded amino acid sequence. Then there are (for further example) sequence variants importing xe2x80x9cconservativexe2x80x9d amino acid changes eg changes from one acidic amino acid to another, one aromatic amino acid to another, one basic amino acid to another, one aliphatic hydrophobic sidechain to another, as is well known in the art. Then there are for example variants corresponding to allelic variants in the encoded polypeptide, and other variants that result in polypeptides of antigenic cross-reactivity and/or similar binding specificity.
The antigenicity of the polypeptides and variants mentioned in the specification includes for example antigenic determinants shared or cross-reactive with the OX-40 antigen as encoded by Seq ID No. 1, eg antigenic polypeptides with determinants that are shared with determinants of OX-40 that are accessible to specific binding agents when the OX-40 is present on a cell surface.
Simply finding a target antigen on a particular cell type does not provide a basis for a therapeutic approach which requires depleting the particular cell type. Thus, many antigens are shed from the cell surface and are not suitable as targets for therapy. A further aspect of this invention is the discovery that a specific binding agent such as an antibody raised against the OX-40 protein and conjugated to a cytotoxin can inhibit the in vitro proliferation of antigen activated CD4+ T-cells. This discovery implies that the OX-40 antigen is rapidly internalized by CD4+ T-cells. Additional research based on this discovery led to an important focus of the present invention; a demonstration that a population of antigen activated CD4+ T-cells can be depleted in vivo by conjugating a specific binding agent such as an antibody raised against the OX-40 antigen with a cytotoxin to produce an immunotoxin, and administering this immunotoxin to a host. In this manner, the antibody binds to the OX-40 antigen on the surface of the activated CD4+ T-cell. Internalization of the immunotoxin results in the cytotoxin being taken into the cell, which produces cell death. Hence, administration of this immunotoxin to a host suffering from activated T-cell (eg CD4+ T-cell) mediated inflammation depletes (or otherwise inactivates) the activated T-cells especially for example activated CD4+ T-cells at the site of inflammation or other sites, leading to amelioration of subsequent inflammation and/or other clinical signs of disease.
A further aspect of the present invention is therefore a method of treating a patient suffering from a condition mediated by activated T-cells e.g. CD4+ T-cells, which comprises administering to the patient an effective amount of a specific binding agent which can specifically bind to a polypeptide as described above, eg an antibody, conjugated with a cytotoxic agent or a radionuclide, and wherein the antibody recognises and binds to the OX-40 antigen present on the surface of the T-cells, especially for example activated CD4+ T-cells.
Particular methods of the invention include a method for reducing a population of T-cells, e.g. CD4+ T-cells that express an OX-40 antigen in a human host and a method of inhibiting relapsing autoimmune inflammation in a patient suffering from multiple sclerosis, which methods comprise administering an effective amount of a specific binding agent of the invention.
In particular the specific binding agent used in these methods is an antibody conjugated with a cytotoxic agent or a radionuclide wherein the antibody recognizes and binds to the OX-40 antigen present on the surface of the T-cells especially for example activated CD4+ T-cells. Alternatively the method may employ a specific binding agent which comprises a Fab, F(abxe2x80x2)2, or Fv fragment of a monoclonal antibody capable of recognising Ox-40 antigen when expressed on the surface of T-cells. Other specific binding agents useful in this method are immunoglobulins capable of cytotoxic effect on cells bearing Ox-40 on their surface or any specific binding agents which can fix, complement or mediate antibody-dependant cellular cytotoxicity such as a specific binding agent which has or is linked to structure characteristic of the Fc region of an immunoglobulin of murine type IgG2a or human type IgG1 or IgG3.
The methods of the invention are applicable to any condition mediated by activated T-cells especially for example activated CD4+ T-cells, including, multiple sclerosis, sarcoidosis, rheumatoid arthritis, autoimmune uveitis, T-cell lymphomas and rejection of a transplanted organ or tissue. Additional conditions to which this method is applicable include graft-versus-host disease or reaction and inflammatory bowel disease.
T-cells can be activated by for example antigens, superantigens, mitogens, or monoclonal antibodies.
The methods of treatment set forth in the preceding paragraph will preferably be performed using specific binding agents such as monoclonal antibodies, or fragments thereof, which can be raised using the polypeptides of the invention. In a more preferred embodiment, the monoclonal antibody will be a humanized monoclonal antibody. In alternative embodiments, the method will utilize a cytotoxic conjugate, eg comprising a fragment such as a Fab, F(abxe2x80x2)2 or Fv fragment of a monoclonal antibody conjugated with a cytotoxic agent wherein the fragment of the monoclonal antibody recognizes the OX-40 antigen.
This invention also encompasses specific binding agents such as monoclonal antibodies having a specificity of binding in cells to substantially only antigen activated T-cells especially for example activated CD4+ T-cells. In a preferred embodiment, the specific binding agents can specifically bind to a polypeptide of the invention. Particular specific binding agents specifically bind to human Ox-40 of amino acid sequence encoded by the coding region of nucleic acid sequence Seq ID No 1, when said Ox-40 is present on the surface of activated cells.
These specific binding agents suitably comprise an antibody binding domain and are preferably monoclonal antibodies or binding fragments thereof. As mentioned above, the antibodies will preferably be at least partially humanised, and so most preferably comprise a humanised monoclonal antibody.
xe2x80x9cSpecific bindingxe2x80x9d refers for example to specific non-covalent molecular binding such as that between an antibody and a corresponding antigen or hapten (its xe2x80x9cbinding partnerxe2x80x9d) and also between other specialised binding molecules and their binding partners.
xe2x80x9cAt least partly humanisedxe2x80x9d, relating to antibodies and their binding domains, means that for example embodiments are contemplated in which only the constant region (CH and CL) may correspond to human polypeptide: alternatively, both the constant and variable regions may be xe2x80x9chumanisedxe2x80x9d.
Another aspect of this invention provides a specific binding agent such as a monoclonal antibody as described above which further comprises a molecularly linked, eg covalently conjugated, cytotoxin. Antibody-cytotoxin conjugates (also known as immunotoxins) are suitable for use in the methods of treatment described above. Examples of such specific binding agents include Fab, F(abxe2x80x2)2 or Fv fragments of a monoclonal antibody conjugated with a cytotoxic agent.
For therapeutic use, the specific binding agents of the invention are suitably administered in the form of a pharmaceutical composition which include a pharmaceutically acceptable carrier. The carrier may be solid or preferably liquid carriers such as water or saline, which are conventional in the art.
A further aspect of the invention comprises a method of detecting a condition mediated by activated T-cells, e.g CD4+ T-cells, in a patient comprising contacting a specific binding agent as described above with said T-cells and quantifying the level of activated T-cells. Suitably the method is carried out on a biopsy sample from the patient, such as a skin or intestinal biopsy sample or a blood sample from a patient suspected of having a graft-versus-host disease, or an intestinal biopsy sample from a patient suspected of having an inflammatory bowel disorder, or a sample of cerebrospinal fluid.
A further aspect of the present invention is a method of detecting an inflammatory condition mediated by activated T-cells especially for example activated CD4+ T-cells in a patient by obtaining a suitable biopsy sample from the patient and then quantifying the percentage of activated T-cells especially for example activated CD4+ T-cells in the biopsy sample using a specific binding agent such as an antibody that specifically binds to the OX-40 antigen. Other aspects of the present invention include test kits for detecting conditions mediated by activated T-cells especially for example activated CD4+ T-celis, and treatment kits comprising antibody in pharmaceutically administrable forms and amounts with suitable excipients and containers.
In particular the invention provides a kit for detecting a condition mediated by activated T-cells e.g. CD4+ T-cells in a patient comprising a specific binding agent as described above which is labelled. Preferably the invention provides a kit for carrying out a specific binding assay for detection or quantitation of an analyte that comprises a polypeptide or a specific binding agent as described above, wherein said kit comprises a first reagent comprising a specific binding agent that can recognise the analyte, a second reagent comprising a substance that can bind specifically either to the analyte or to the first reagent, and a label for the second reagent.
In such a kit the first reagent can comprise an antibody specific for the analyte, and the second reagent comprises a labelled antiglobulin specific for the first reagent.
Alternatively the first reagent can comprise an immobilised specific binding reagent for the analyte, then the second reagent is a specific binding agent that can bind to the analyte when the analyte is also bound to the first reagent.
In a further embodiment wherein the second reagent comprises a substance able to compete with analyte for binding to the first reagent.
For use in this method, the specific binding agent may further comprises a label such as a radioactive label or a fluorescent label and these specific binding agents form a further aspect of the invention.
The specific binding agents of the invention may be cloned and sequenced in the usual way. Thus the invention further provides a nucleic acid sequence encoding the amino acid sequence of a specific binding agent as described above.
Recombinant technology may be used to prepare both the polypeptides and the specific binding agents of the invention. Thus appropriate expression vectors, transformed host organisms and methods of preparation which include culturing a host organism form further aspects of the invention.
The OX40 binding agent, for instance an anti-OX40 monoclonal antibody, can be administered to those patients suffering from a disease mediated by activated T cells e.g. graft-versus-host disease. The amount administered will depend on the amount required to produce an improvement, either partial or total, in the patient""s symptoms. This will depend not only on the severity of the condition and route of administration but also on the administration of other therapeutic agents (eg glucocorticoids, cyclosporine A, prednisolone). The OX40 binding agent may be injected either systemically (eg intravenous) or locally (eg intramuscular). As discussed elsewhere the OX40 binding agent may be coupled to a toxic substance for maximum therapeutic effect.
OX-40 and Graft Versus Host Disease (GVHD)
In the case of GVHD, immunocompetent T cells derived from the donor tissue or cells attack recipient tissue including skin, gut and liver, which are severely compromised in their ability to carry out their normal function. Such attacks, if not controlled, can lead to death of the patient. The therapeutic agents of the present invention could be used to block the activation of or to eliminate the donor T cells thereby preventing or halting the disease process.
OX-40 and GvHD: Diagnostics
The present invention can be used in diagnostic tests and procedures in vitro. For instance the OX40 binding agent can be used to determine the presence of OX40+ T cells in a biopsy sample from a patient. The biopsy sample may be a tissue sample or a sample of blood. Mononuclear cells are isolated from the blood or tissue according to standard techniques (see Practical Immunology. L. Hudson and F. C. Hay, eds. Blackwell Scientific Publications, Oxford) and stained with an anti-OX40 antibody or OX40 binding agent fusion protein. The presence of the OX40 binding agent is then detected with an anti-globulin reagent coupled with a fluorochrome such as fluoroscein isothiocyanate or phycoerythrin and the number of positive cells analysed on a flow cytometer or by fluorescence microscopy (see eg Practical Flow Cytometry. Shapiro, H. M., ed. Alan R Liss, New York; Practical Immunology. L. Hudson and F. C. Hay, eds. Blackwell Scientific Publications, Oxford). Alternatively the tissue sample is processed for immunohistochemical staining by standard techniques (see eg Immunocytochemistry: Practical Applications in Pathology and Biology. J. Polak and S. van Noorden, eds. John Wright and Sons, Bristol). The OX40+ population of cells could be further characterised by two- or three-colour flow cytometry or immunohistochemistry (see eg Practical Flow Cytometry. Shapiro, H. M., ed. Alan R. Liss, New York; Immunocytochemistry: Practical Applications in Pathology and Biology. J. Polak and S. van Noorden, eds. John Wright and Sons, Bristol). Detection of OX40+ cells can aid in the diagnosis and management of diseases caused by activated T cells e.g. inflammatory bowel disease and GvHD and may be used to follow the course of the disease: an increase in the proportion of OX40+ cells would suggest a worsening of the disease and may indicate the need to increase the dose of therapeutic agent being administered, while a decrease in the proportion of OX40+ cells would suggest an improvement and thus indicate a diminution in the amount of therapeutic agent being administered.
Determination of the levels of OX40+ cells in the blood of patients at risk of GvHD (eg following allogenic bone-marrow transplantation) may allow one to predict the imminent onset of GvHD. Early administration of immunosuppressive agents to control GvHD will improve the likelihood of successful treatment.
The present invention can be used in diagnostic tests and procedures in vivo. For instance, the administration of an OX40 binding agent coupled to a radioisotope can be used for the purposes of immunoscintigraphy.
These and other aspects of the present invention will become more readily apparent from the following figures and description of the invention.